RNA interference or “RNAi” is a term coined by Fire and co-workers to describe the observation that certain double-stranded RNAs (dsRNAs) blocked gene expression when they were introduced into worms. Introduction of dsRNA into a cell leads to the sequence-specific destruction of endogenous RNAs that have sequences that are complementary to the dsRNA. During RNAi, long dsRNA molecules are processed into 19- to 23-nt RNAs known as short-interfering RNAs (siRNAs) that serve as guides for enzymatic cleavage of complementary RNAs. In addition, siRNAs can function as primers for an RNA-dependent RNA polymerase that synthesizes additional dsRNA, which in turn is processed into siRNAs, amplifying the effects of the original siRNAs. In mammalian cells, dsRNA is processed into siRNAs, but RNAi with dsRNA has not been successful in most cell types because of nonspecific responses elicited by dsRNA molecules longer than about 30 nt. However, Tuschl and coworkers observed that transfection of synthetic 21-nt siRNA duplexes into mammalian cells effectively inhibited endogenous genes in a sequence-specific manner. These siRNA duplexes are too short to trigger the nonspecific dsRNA responses, but they still cause destruction of complementary RNA sequences.
One particularly problematic aspect of administering any pharmaceutical compound is the delivery of the compound to the desired tissue in the patient. In particular, antisense and siRNA therapeutics to date have been hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. For siRNA therapeutics for cancer, the pharmacological hurdles are severe. Local aqueous siRNA activity has been observed for several tissues, but is lacking in tumors, and systemic exposure can induce non-specific responses, as found for CpG DNA oligonucleotides. Although the potency and selectivity of siRNA inhibitors of gene expression promises to enable improved targeted cancer therapeutics, the means for systemic administration and targeted distribution to affected cells and tissues are needed.
Two basic approaches for targeting tumors with the avidin-biotin system have been used in patients and animals. In the first method, avidin (or streptavidin)-conjugated antibodies are injected and days later when antibody-tumor binding is maximized, a radioactive biotin derivative is injected to localize the tumor. Unfortunately, incomplete clearance of unbound antibody from the blood can obscure visualization of the target site. In the second method, blood background is reduced by injecting biotinylated antibodies followed three days later by cold avidin. The resultant circulating biotinylated antibody-avidin complexes are sequestered from the blood by the liver. Radioactive biotin is then injected which binds to the antibody-biotin-avidin complexes already localized in the tumor. However, by employing “pretargeting” steps, both approaches for targeting tumors require that a subject be available to undergo multiple procedures over the course of a few days.